Embryonic Stem Cell Pluripotency and Early Differentiation

干细胞代表了再生医学的明显选择，对人类发育和毒理学的研究是无价的。特别是多能干细胞（PSCs）的蛋白质组学景观尚未明确定义；因此，这一领域的研究将大大受益于旨在更好地表征这些细胞群的协同努力。最近，我们开始对干细胞的潜能进行概述，强调了PSCs异质性的类型和实际意义，并对多能性蛋白质组在一种独特的、快速进化的资源中的当前观点进行了详细的分析。我们的目标是为小鼠和人类psc的已知蛋白质组的现状提供具体的见解。这是通过将已发表的数据整合到统一的PSC蛋白质组中来完成的，以促进对干细胞状态提供信息的蛋白质的鉴定，并揭示我们目前观点有限的领域。这些分析提供了对PSCs蛋白质组学分析面临的挑战的见解，并揭示了细胞表面亚蛋白质组学的一个领域，特别是从加强研究工作中受益。

Stem cells represent obvious choices for regenerative medicine and are invaluable for studies of human development and toxicology. The proteomic landscape of pluripotent stem cells (PSCs), in particular, is not yet clearly defined; consequently, this field of research would greatly benefit from concerted efforts designed to better characterize these cell populations. Recently, we have begun to develop an overview of stem cell potency, highlight the types and practical implications of heterogeneity in PSCs and provide a detailed analysis of the current view of the pluripotent proteome in a unique and rapidly evolving resource. Our goal has been to provide specific insights into the current status of the known proteome of both mouse and human PSCs. This has been accomplished by integrating published data into a unified PSC proteome to facilitate the identification of proteins which may be informative for the stem cell state as well as to reveal areas where our current view is limited. These analyses provide insight into the challenges faced in the proteomic analysis of PSCs and reveal one area the cell surface subproteome that would especially benefit from enhanced research efforts. More recently, we have begun a targeted identification of plasma membrane N-glycoprotein extracellular domains by Cell Surface Capturing Technology revealed 500 cell surface accessible proteins on mouse embryonic and induced pluripotent stem cells, including 187 not previously reported in mouse pluripotent stem cells. This new resource includes 91 CD molecules and represents a functional surface molecule "barcode" for the pluripotent state. Emerging from this "barcode" are informative markers for the pluripotent state that can be used for sorting live subpopulations of PSCs, including isolating iPSCs from heterogeneous mixture of reprogramming cells. The results from this targeted strategy represent an important stem cell resource that will accelerate the development of surface protein markers and affinity reagents for immunophenotyping and isolating pluripotent stem cells. These data from pluripotent mouse and human ESCs have been recently published. Finally, we have extended our previous work on B-MYB and its role in undifferentiated stem cells. We have found that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain self-renewal of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells, and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we have unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases as well as pluripotency transcription factors, epigenetic regulators, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. Meanwhile B-MYB together with OCT4, SOX2, and NANOG co-target genes significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB actively up-regulates factors critical to self-renewal, including key regulatory proteins important for cell cycle progression and fate decisions. These data were also recently published.